Epithelial cells require regulated vesicle trafficking in order to maintain their dynamic actin cytoskeleten and polarity. Although many signaling pathways have been studied, how a cell coordinates the regulation of membrane-trafficking and cytoskeletal organization remains unclear. Recent work from our laboratory has revealed that a microvillar protein, EPI64, has regulatory functions for both Arf6 and an unknown Rab protein. EPI64's TBC domain binds directly to active Arf6-GTP and EPI64's over- expression induces an accumulation of actin-coated vacuoles likely driven by the over production of PIP2 via PI4P5K activation. Expression of a GAP-defective mutant that still binds to Arf6-GTP, EPI64_R160A, fails to induce vacuoles, indicating that GAP activity is required for vacuole accumulation. Additionally, the appearance of these vacuoles by EPI64 expression is eliminated by the co-expression of Rab8a. These results suggest that EPI64 coordinates Arf6 activity with a Rab protein, possibly Rab8, in a novel manner to regulate both membrane traffic and cytoskeletal organization. The goal of this research is to identify the relevant Rab protein(s) for EPI64's GAP activity, and to elucidate how EPI64 participates in these trafficking pathways. The first aim will be to identify which Rab proteins are affected by the overexpression of EPI64. Since overexpression of EPI64 results in the formation of actin-coated vacuoles in the cell, this can be used as an assay to screen for Rab proteins that are able to overwhelm the GAP activity of EPI64, and therefore be the relevant regulator of the recycling pathway. The biochemistry of Rab proteins potentially relevant to EPI64 will be studied in Aim 2 by measuring Rab-GTP levels in the cell using effectors in a pull-down assay. GAP activity of EPI64 on the Rab proteins found to have EPI64 dependent changes in Rab-GTP levels will be quantitatively measured using purified proteins. Aim 3 will focus on determining the formation and structure of the vacuoles that form in the cell when these membrane-trafficking pathways are perturbed. High resolution immunofluorescence live-cell imaging will be used to determine the temporal and spatial co- localization of GFP-tagged molecules during actin-coated vacuole formation. This will be complemented by transmission electron microscopy to image the formation and structure of vacuoles at various time points. Relevance: Maintaining a distinct polarity in epithelial cells that form the lining of internal structures in the intestine or kidney is vital for the uptake and distribution of nutrients. Disrupting the intracellular trafficking networks of these cells can lead to malnutrition, cancer, and eventual death.